Protein Assay

ABSTRACT

A method is described for the measurement of thrombin activity in the presence of fibrinogen, or for the measurement of the functionality of fibrinogen in the presence of thrombin.

FIELD OF THE INVENTION

A method is described for the measurement of thrombin activity in thepresence of fibrinogen, or for the measurement of the functionality offibrinogen in the presence of thrombin.

BACKGROUND OF THE INVENTION

Fibrinogen and thrombin are critical proteins involved in achievinghemostasis after vascular injury and essential to blood clot formation.Fibrinogen and thrombin can be combined in powder form or in anon-aqueous suspension, without initiating a typical clotting reaction,thus preventing the formation of a fibrin clot until the proteins arehydrated in an aqueous medium or other liquid environment in which theproteins are soluble. An admixture of these proteins in powder form havea variety of potential biomedical applications including topicalhemostasis, tissue repair, drug delivery, etc. In addition, an admixtureof these proteins may be loaded onto a carrier or substrate, or othermedical device, in powder form to form a product that may be used forexample as a hemostatic device.

The clotting activity of thrombin is usually measured by combiningthrombin in solution with a known amount of fibrinogen in solution. Withappropriate conditions, the rate of clot formation after combining thetwo proteins is dependent on the activity of the thrombin. The rate ofclot formation of a sample with an unknown amount of thrombin iscompared with the rate of clot formation of a thrombin reference orthrombin standard to determine the activity of the sample.

Thrombin activity is a critical attribute of any thrombin/fibrinogenproduct and will dictate its functionality. Although the measurement offree thrombin is straightforward, measurement of thrombin activity whenthrombin and fibrinogen are in an unreacted admixture has been achallenge since measurement thereof typically requires that theadmixture of proteins be hydrated and solubilized, and fibrin clotformation between solubilized thrombin and fibrinogen immediatelyinitiates upon hydration. Furthermore, since thrombin is known to bindand interact specifically with the immediately formed fibrin clot, thethrombin becomes bound in the fibrin clot and is no longer freelysoluble in the hydrating solution and becomes unavailable for subsequentmeasurement of thrombin activity. Hence, any resultant measurement ofthe thrombin activity of any thrombin/fibrinogen product via hydrationand clot formation is only partial and hence inaccurate.

Moreover, when the proteins are in an unreacted admixture and loadedonto a carrier, substrate or medical device, it may be necessary toremove the proteins from the substrate to accurately measure thethrombin activity, for example, if the carrier, substrate or medicaldevice adversely affects the measurement of the activity orfunctionality of the proteins due to physical, chemical or opticalinterference with the measurement detection system. To overcomeinterference from the carrier, substrate or medical device, removal orextraction of the proteins must be performed without exposing theadmixture to aqueous conditions, which would result in clot formationpreventing subsequent measurement.

Fibrinogen is most frequently measured by a method originally describedby Clauss, which measures fibrinogen functionality based on the rate ofclot formation. In a typical Clauss assay, a sample with an unknownamount of soluble fibrinogen is combined with an excess of thrombin. Theproportions of fibrinogen and thrombin are such that fibrinogen is therate limiting reactant and the rate of clot formation is a function ofthe fibrinogen concentration. A fast clotting time would be indicativeof a high fibrinogen concentration. Conversely, a longer dotting timewould indicate a low concentration of functional fibrinogen. The amountof functional fibrinogen can be quantified by comparing the clottingtime of the sample with those of a series for standards to establish astandard curve. The concentration of fibrinogen in the sample can bedetermined mathematically based on the equation derived from theclotting times of the standards.

While measurement of free fibrinogen in solution. e.g., human plasma,can be performed by established methods, assessment of fibrinogenfunctionality when fibrinogen is in the presence of thrombin has been achallenge. Hydration of the admixture will result in thrombin-mediatedconversion of fibrinogen into an insoluble fibrin clot. Once fibrin isgenerated, any subsequent measurement of fibrinogen is no longerpossible since the release of the fibrinopeptides from fibrinogenresulting in fibrin formation is essentially irreversible.

Hence there remains a need to accurately measure the activity ofthrombin in the presence of fibrinogen, and to measure the functionalityof fibrinogen in the presence of thrombin.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the influence of pH of the inactivation solution onrecovered thrombin activity.

SUMMARY OF THE INVENTION

Described herein is a method for determining the activity orfunctionality of either a first reactive component or a second reactivecomponent in an admixture of the first reactive component and the secondreactive component, comprising the steps of (a) reversibly inhibitingthe first reactive component to yield a mixture having an inactivatedfirst reactive component and the second reactive component; (b) addingto the mixture an excess of the second reactive component whenevaluating the activity of the first reactive component, or an excess ofthe first reactive component when evaluating the activity of the 30second reactive component; (c) reversibly activating the first reactivecomponent; (d) allowing the first reactive component to react with thesecond reactive component in the mixture and the excess of the secondreactive component, or allowing the first reactive component to reactwith the second reactive component in the mixture and the excess of thefirst reactive component; and (e) determining the activity orfunctionality of first or second reactive component originally presentin the dry mixture.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, in order to determine the thrombin activity of anunreacted admixture of thrombin and fibrinogen, for example in powderform or a non-aqueous suspension such as an ethanol suspension, it isnecessary to rehydrate the proteins and for the thrombin and fibrinogento be solubilized in the hydrating medium, to obtain an accuratemeasurement of the thrombin activity. However, once the admixture iscontacted with the hydrating medium, any solubilized thrombin andfibrinogen will react to form an immediate clot, and any availablethrombin will bind to the clot and will not be freely available for themeasurement thereof.

In one embodiment, the thrombin activity of the unreacted admixture istemporarily inhibited or reversibly inhibited, thereby preventing theformation of a fibrin clot until the thrombin and fibrinogen arecompletely solubilized. By inhibiting thrombin activity, immediate clotformation is avoided and the thrombin is able to freely dissolve in anaqueous hydrating medium and remains available for measurement.

The temporary or reversible inhibition of thrombin activity can beachieved, for example, by adjusting the alkaline environment of thethrombin. For example, this may be accomplished by reconstituting orhydrating an unreacted admixture of thrombin and fibrinogen in aninhibitory or inactivation solution, i.e., an alkaline solution having apH ranging from about 8.5 to 11.5, preferably from about 9.5 to 10.5,and more preferably about 10, to form a first solution. Table 1 showsthe effect of pH on the recovered activity of thrombin. Maximalrecovered thrombin activity was observed when the alkalinity of theinactivation solution was at pH 10. Within a pH range of 9.5-10.5, atleast 80% of the maximal recovered thrombin activity was achieved. At pHlevels less than 9.5 and greater than 10.5, the maximal recoveredthrombin activity decreased as the pH level deviated further from 10. AtpH levels of 9.25 and lower, evidence of clot formation was observedduring hydration and may explain the reduced maximal recovered thrombinactivity that is observed at lower pH values approaching neutralconditions. In acidic conditions, for example of pH 4 and 5, maximalrecovered thrombin activity was significantly less than that observedwith alkaline conditions, which may be an indication of the irreversibleinactivation of the thrombin.

The inhibitory or inactivation solution may be an alkaline solution or abuffered alkaline solution, including but not limited to a solution ofcarbonate, TRIS base, borate, glycine, phosphate, methylamine,2-(Cyclohexylamino)ethanesulfonic acid (CHES),3-(Cyclohexylamino)-1-propanesulfonic acid (CAPS) or3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO).

Once the thrombin and fibrinogen are completely solubilized in theinhibitory or inactivation solution, the first solution or a portionthereof may be combined with a known amount of fibrinogen in a secondsolution, preferably having an excess amount of fibrinogen to form athird solution, while maintaining the pH at about 8.5 to 11.5,preferably from about 9.5 to 10.5, and more preferably about 10. Anexcess of fibrinogen is utilized so that the amount of thrombin in theadmixture is the rate limiting reactant in the formation of the fibrinclot, to ensure that the activity of thrombin correlates strongly withthe rate of clot formation. If fibrinogen is not in excess, the rate ofclotting would be dependent upon both thrombin and fibrinogen.

Thereafter, the thrombin activity may be reversed, for example, byadjusting the pH of the third solution to a range where the thrombinactivity is no longer inhibited, i.e., from about 6.0 to less than 8.5,preferably from about 7.0 to less than 8.5, and more preferably about7.5. Alternatively, the inactivation solution having the solubilizedproteins or a portion thereof, i.e., the first solution, may be combinedwith an known amount of fibrinogen in a second solution, preferably anexcess amount of fibrinogen, to form a third solution whereby theinhibition of the thrombin activity is simultaneously reversed. Examplesof the second solution include but are not limited to buffer solutionsfor TRIS-HCl, imidazole, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonicacid (HEPES), phosphate, barbital, 4-Morpholinepropanesulfonic acid(MOPS), 3-Morpholino-2-hydroxypropanesulfonic acid (MOPSO),1,4-Piperazinediethanesulfonic acid (PIPES),N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), citrate orcarbonate.

The volume and buffering capacity of the second solution should besufficient to result in a third solution having a pH from about 6.0 toless than 8.5, preferably from about 7.0 to less than 8.5, and morepreferably about 7.5, when added to the first solution. For example, theratio of the volumes of the first and second solutions typically rangesfrom about 1:1 to 1:20, and preferably is about 1:4 to 1:10, forexample, when the molarity of the second solution is about 25 mM to 500mM TRIS-HCl buffer, and preferably about 100 mM to 150 mM TRIS-HClbuffer.

The thrombin activity may be determined using a coagulation analyzerwith a mechanical endpoint detection system to detect clot formation,such as the Diagnostica Stago ST4 Coagulation Analyzer, or a device thatmeasures changes in turbidity due to fibrin clot formation. Thesolubilized proteins in the inactivation solution may be combined withthe second solution in one of these devices, and the time to coagulationmay be measured, which can then be correlated to the clotting times forknown thrombin activities.

Another method by which thrombin activity can be measured is using achromogenic or fluorogenic peptide substrate for thrombin. In thismethod, solubilized thrombin is combined with an excess of chromogenicor fluorogenic substrate. Thrombin will cleave the substrate releasing achromophore or fluorophore which can be monitored in a spectrophotometeror fluorimeter. Examples of chromogenic or fluorogenic substratesinclude, 3-Ala-Gly-Arg-p-nitroanilide diacetate and Z-Gly-Pro-Arg-AMC[Z=Benzyloxycarbonyl; AMC=7-amino-4-methylcoumarin], respectively. Therate of released chromophore or fluorophore can be correlated to theactivity of thrombin.

In another embodiment, the functionality of the fibrinogen in anunreacted admixture with thrombin may be measured by inhibiting thethrombin activity by adjusting the alkaline environment of the thrombin.For example, this may be accomplished by reconstituting or hydrating anadmixture of thrombin and fibrinogen in an inhibitory or inactivationsolution, i.e., an alkaline solution having a pH ranging from about 8.5to 11.5, preferably from about 9.5 to 10.5, and more preferably about10, to form a first solution. The inhibitory or inactivation solutionmay be an alkaline solution or a buffered alkaline solution, includingbut not limited to a solution of carbonate,TRIS(Tris(hydroxymethyl)aminomethane) base, borate, glycine, phosphate,methylamine, 2-(Cyclohexylamino)ethanesulfonic acid (CHES),3-(Cyclohexylamino)-1-propanesufonic acid (CAPS) or3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO).Additionally and optionally, a thrombin inhibitor such as Bivalirudin(Angiomax) may be added to the inhibitory or inactivation solution orthe first solution to achieve maximum inhibition of thrombin activitythus allowing most of the fibrinogen to be solubilized for subsequenttesting. Other examples of thrombin inhibitors include antithrombin,heparin, low molecular weight heparin, low molecular weight heparinanalogs, argatroban, melagatran, efegatran, inogatran, dabigatran,hirudan and derivatives of hirudan such as Lepirudin, and Desirudin.

Once the thrombin activity has been inhibited, the functionality offibrinogen can be determined by combining the first solution or aportion thereof with a known amount of thrombin in a second solution,preferably having an excess amount of thrombin to form a third solution,while maintaining the pH at about 8.5 to 11.5, preferably from about 9.5to 10.5, and more preferably about 10. An excess of thrombin is utilizedso that the amount of fibrinogen in the admixture is the rate limitingreactant in the formation of the fibrin dot, to ensure that theconcentration of fibrinogen correlates strongly with the rate of clotformation. If thrombin is not in excess, the rate of clotting would bedependent upon both thrombin and fibrinogen.

Thereafter, the thrombin activity may be reversed, for example, byadjusting the pH of the third solution to a range where the thrombinactivity is no longer inhibited, i.e., from about 6.0 to less than 8.5,preferably from about 7.0 to less than 8.5, and more preferably about7.5. Alternatively, the inactivation solution having the solubilizedproteins or a portion thereof, i.e., the first solution, may be combinedwith an known amount of thrombin in a second solution, preferably anexcess amount of thrombin, to form a third solution whereby theinhibition of the thrombin activity is simultaneously reversed. Examplesof the 10 second solution include but are not limited to buffersolutions for TRIS-HCl, imidazole,4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), phosphate,barbital, 4-Morpholinepropanesufonic acid (MOPS),3-Morpholino-2-hydroxypropanesulfonic acid (MOPSO),1,4-Piperazinediethanesulfonic acid (PIPES),N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), citrate orcarbonate at pH of about 7.5.

The functionality of the fibrinogen may be determined using acoagulation analyzer with a mechanical endpoint detection system todetect clot formation, such as the Diagnostica Stago ST4 CoagulationAnalyzer, or a device that measures changes in turbidity due to fibrinclot formation. The solubilized proteins in the inactivation solutionmay be combined with the second solution in one of these devices, andthe time to coagulation may be measured, which can then be correlated tothe clotting times for known fibrinogen functionalities.

Alternatively, the fibrinogen functionality may be determined byinhibiting the thrombin activity using a thrombin inhibitor such asBivalirudin (Angiomax). Optionally, the alkaline environment of thethrombin may be adjusted in combination with the use of the thrombininhibitor. Other examples of thrombin inhibitors include antithrombin,heparin, low molecular weight heparin, low molecular weight heparinanalogs, argatroban, melagatran, efegatran, inogatran, dabigatran,hirudan and derivatives of hirudan such as Lepirudin, and Desirudin.Once the thrombin activity is inhibited, the fibrinogen functionalitymay be determined by using a thrombin-like enzyme that is capable ofacting on fibrinogen to form a clot, but is unaffected by the thrombininhibitor. Examples of thrombin-like enzymes include but are not limitedto Batroxobin (derived from the venom of the South American pit viperBothrops atrox) and Ancrod (derived from the venom of Calloselasmarhodostoma).

In the event the proteins are in an unreacted admixture and loaded ontoa carrier, substrate or medical device, for example, the admixture maybe in powder form where the proteins are dry or dried, removal of theproteins prior to rehydration and solubilization may be performed byextracting the proteins using a non-aqueous liquid, including but notlimited to perfluorinated hydrocarbons such as HFE-7000, HFE7001,HFE7003, HFE-7300 and PF-5060 (commercially available from 3M ofMinnesota) and any other carrier fluid in which the proteins do notdissolve may be used, such as alcohols, ethers or other organic fluids.Once the proteins have been extracted using the non-aqueous solvent, thethrombin activity or fibrinogen functionality may be measured asdescribed above.

Alternatively, where the proteins are loaded onto a carrier, substrateor medical device, the thrombin activity or fibrinogen functionality maybe determined as described above without removal of the proteins. Forexample, the proteins may be hydrated by placing the carrier, substrateor medical device having the proteins thereon directly in the inhibitoryor inactivation solution which can be sampled for testing for thrombinactivity or fibrinogen functionality as described above.

We claim:
 1. A method for determining the activity or functionality ofeither a first reactive component or a second reactive component in anunreacted admixture of the first reactive component and the secondreactive component, comprising the steps of (a) reversibly inhibitingthe first reactive component to yield a mixture having an inactivatedfirst reactive component and the second reactive component; (b) addingto the mixture a known amount of the second reactive component whenevaluating the activity of the first reactive component, or a knownamount of the first reactive component when evaluating the activity ofthe second reactive component; (c) reversibly activating the firstreactive component; (d) allowing the first reactive component to reactwith the second reactive component originally present in the admixtureand the known amount of the second reactive component, or allowing thefirst reactive component to react with the second reactive componentoriginally present in the admixture and the known amount of the firstreactive component; and (e) determining the activity or functionality offirst or second reactive component originally present in the admixture.2. The method of claim 1, where the first reactive component is thrombinand the second reactive component is fibrinogen.
 3. The method of claim2, where the thrombin is recombinant or derived from an animal or humansource, and the fibrinogen is recombinant or derived from an animal orhuman source.
 4. The method of claim 2, where steps (b) and (c) areperformed simultaneously.
 5. The method of claim 4, where the admixtureis in powder form and the step of reversibly inhibiting the firstreactive component comprises reconstituting the admixture of the firstreactive component and a second reactive component in an inhibitory orfirst solution having a pH ranging from 8.5 to 11.5.
 6. The method ofclaim 5, where the inhibitory or first solution has a pH of 9.5 to 10.5.7. The method of claim 6, where the inhibitory or first solution has apH of about
 10. 8. The method of claim 5, where the inhibitory or firstsolution comprises an alkaline solution of at least one componentselected from the group consisting of carbonate, TRIS(Tris(hydroxymethyl)aminomethane) base, borate, glycine, phosphate,methylamine, 2-(Cyclohexylamino)ethanesulfonic acid (CHES),3-(Cyclohexylamino)-1-propanesufonic acid (CAPS) or3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO).
 9. Themethod of claim 8, where the known amount of the second reactivecomponent is in a second solution that is capable of lowering the pH ofthe inhibitory or first solution having the inactivated first reactivecomponent and the second reactive component to about 6.0 to less than8.5.
 10. The method of claim 9, where the known amount of the secondreactive component is in a second solution that is capable of loweringthe pH of the inhibitory or first solution having the inactivated firstreactive component and the second reactive component to about 7.0 toless than 8.5.
 11. The method of claim 10, where the known amount of thesecond reactive component is in a second solution that is capable oflowering the pH of the inhibitory or first solution having theinactivated first reactive component and the second reactive componentto about 7.5.
 12. The method of claim 9, where the second solutioncapable of lowering the pH of the inhibitory or first solution is asolution of at least one component selected from the group consisting ofTRIS-HCl, imidazole, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid(HEPES), phosphate, barbital, 4-Morpholinepropanesulfonic acid (MOPS),3-Morpholino-2-hydroxypropanesutfonic acid (MOPSO),1,4-Piperazinediethanesulfonic acid (PIPES),N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), citrate orcarbonate.
 13. The method of claim 12, where the second solution capableof lowering the pH of the inhibitory or first solution comprises 25 mMto 500 mM TRIS-HCl buffer.
 14. The method of claim 13, where the secondsolution capable of lowering the pH of the inhibitory or first solutioncomprises 100 mM to 150 mM TRIS-HCl buffer.
 15. The method of claim 9,where the known excess amount of the second reactive component is addedto the inhibitory or first solution having an inactivated first reactivecomponent and the second reactive component in step (b).
 16. The methodof claim 2, where step (b) comprises adding a second solution having aknown amount of the second reactive component to the inhibitory or firstsolution having the inactivated first reactive component and the secondreactive component to form a third solution having a pH ranging from 8.5to 11.5.
 17. The method of claim 16, where step (c) of reversiblyactivating the first reactive component comprises adjusting the pH ofthe third solution to about 6.0 to 8.5.
 18. The method of claim 9, wherethe activity or functionality of the first or second reactive componentoriginally present in the admixture is determined by a kinetic clottingassay that measures a coagulation time, and the coagulation time iscorrelated to known activities or functionalities of the first or secondactive component.
 19. The method of claim 18, where the activity orfunctionality of the first or second active component is converted toactivity units or functional quantity.
 20. The method of claim 9, wherethe activity or functionality of the first or second reactive componentoriginally present in the admixture is determined using a turbidimetry,nephelometry, or viscomerty, or mechanical endpoint analysis method. 21.The method of claim 2, where the admixture of the first reactivecomponent and a second reactive component is on a substrate in powderform and step (b) is carried out by placing the substrate having theadmixture of the inactivated first reactive component and the secondreactive component in a second solution having a known amount of secondreactive component.
 22. The method of claim 2, where the admixture ofthe first reactive component and the second reactive component is anon-aqueous suspension.
 23. The method of claim 22, where thenon-aqueous suspension comprises alcohol, thrombin and fibrinogen. 24.The method of claim 23, where the alcohol is ethanol.
 25. A method fordetermining the functionality of fibrinogen in an unreacted admixture ofthrombin and fibrinogen, comprising the steps of (a) inhibiting thethrombin to yield a mixture having inactivated thrombin and fibrinogenby reconstituting the admixture of the thrombin and fibrinogen in aninhibitory or first solution having a pH ranging from 8.5 to 11.5; (b)adding to the mixture a known amount of thrombin in a second solutionthat is capable of lowering the pH of the inhibitory or first solutionhaving the fibrinogen and inactivated thrombin to about 6.0 to less than8.5; (c) allowing the thrombin to react with the fibrinogen; and (d)determining the functionality of fibrinogen originally present in theadmixture.
 26. The method of claim 25, where the step of inhibiting thethrombin further comprises adding a thrombin inhibitor.
 27. The methodof claim 26, where the thrombin inhibitor is selected from the groupconsisting of antithrombin, heparin, low molecular weight heparin, lowmolecular weight heparin analogs, fondaparinux, argatroban, melagatran,efegatran, inogatran, dabigatran, bivalirudin, hirudan and derivativesof hirudan such as Lepirudin, and Desirudin.
 28. A method fordetermining the functionality of fibrinogen in an unreacted admixture ofthrombin and fibrinogen, comprising the steps of (a) inhibiting thethrombin to yield a mixture having inactivated thrombin and fibrinogenby reconstituting the admixture of the thrombin and fibrinogen in aninhibitory or first solution having a pH ranging from 8.5 to 11.5; (b)adding to the mixture a known amount of a thrombin-like enzyme andoptionally lowering the pH of the inhibitory or first solution havingthe fibrinogen and inactivated thrombin to about 6.0 to less than 8.5;(c) allowing the thrombin-like enzyme to react with the fibrinogen; and(d) determining the functionality of fibrinogen originally present inthe admixture.
 29. The method of claim 28, where the step of inhibitingthe thrombin further comprises adding a thrombin inhibitor.
 30. Themethod of claim 28, where the thrombin inhibitor is selected from thegroup consisting of antithrombin, heparin, low molecular weight heparin,low molecular weight heparin analogs, fondaparinux, argatroban,melagatran, efegatran, inogatran, dabigatran, bivalirudin, hirudan andderivatives of hirudan such as Lepirudin, and Desirudin.
 31. The methodof claim 28, where the thrombin-like enzyme is Batroxobin or Ancrod. 32.A method for determining the activity of thrombin in an unreactedadmixture of thrombin and fibrinogen, comprising the steps of: (a)reversibly inhibiting the thrombin to yield a mixture having inactivatedthrombin and fibrinogen by reconstituting the admixture of the thrombinand fibrinogen in an inhibitory or first solution having a pH rangingfrom 8.5 to 11.5; (b) adding a second solution that is capable oflowering the pH of the inhibitory or first solution to about 6.0 to lessthan 8.5; (c) allowing the fibrinogen to react with the thrombin; and(d) assessing dot formation.
 33. A method for determining the activityof thrombin in an unreacted admixture of thrombin and fibrinogen,comprising the steps of (a) reversibly inhibiting the thrombin to yielda mixture having an inactivated thrombin and fibrinogen; (b) adding tothe mixture a known amount of a chromogenic or fluorogenic thrombinsubstrate; (c) reversibly activating the thrombin; (d) allowing thethrombin to react with the chromogenic or fluorogenic thrombinsubstrate; (e) determining the activity the thrombin originally presentin the admixture.